Extraction of Artemisinin from Artemisia Annua

Table of Contents

Acknowledgements

List of Tables

List of Figures

List of Acronyms

Abstract

1.Introduction
1.1 Background
1.2 Objective
1.2.1 General objective
1.2.2 Specific Objectives
1.3 Scope of Study

2. Literature Review
2.1 Essential oil utilization and application
2.2 History of Artemisia annua L
2.3 Artemisia Plant and Production
2.4 Uses of Artemisia annua L
2.4.1 Medicinal Uses
2.4.2 Edible Uses
2.5 Chemical Constituents of Artemisia Annua
2.6 Physical and Chemical properties of Artemisia Annua
2.6.1 Physical Properties of Artemisia annua
2.6.3 Physico-Chemical Properties
2.6.4 Over View of Artemisinin Oil
2.7 Composition of Artemisinin Benefits of Artemisinin
2.8 Market Assessment

3. Process technology
3.1 Technology choices
3.2 Process description
3.2.1 Artemisinin extraction by using hexane extraction process
3.3 Process Flow Sheet

4. Materials and Methods
4.1 General description of the study area
4.2 Material Used
4.2.1 Plant Materials
4.2.2 Chemicals
4.3 Laboratory Work
4.3.1 Objective of the Laboratory
4.3.2 Equipment Setup
4.3.3 Laboratory Procedure
4.3.4 Laboratory Findings

5. Large Scale Production
5.1 Comparison of Small Scale and Large Scale Processes

6. Material and Energy Balance
6.1Material Balance
6.2 Energy balance
6.2.1 General Energy Balance

7. Mechanical Equipment Design (Extractor Design)
7.1 Searching for the Right Kind of Large Scale Extractor
7.2 Features
7.3 Specifications
7.4 Design Procedures
7.5 Skirt Design
7.6 Bolt Design
7.7 Reinforcement Calculation

8. Preliminary Equipment Design (Sizing)

9.Economic Analysis
9.1 Purchased Equipment Cost and Cost Estimation
9.2 Profitability Analysis
9.3 Return on Investment (ROI)
9.4 Payback Period
9.5 Net present Worth (NPW)

10. Conclusion and Recommendation
10.1 Conclusion
10.2 Recommendation

References

Appendices

Appendix A: Additional Physico-Chemical Properties of Artemisinin (Malcolm Cutler et al., 2006) 71

Appendix B: Typical artemisinin buyer specification (Malcolm Cutler et al., 2006) 72

Appendix C: Safety Data on n-hexane 73

List of Tables

Table 2.1 Artemisinin content in different samples of A. annua % dry weight

Table 2.2 Scientific classifications

Table 2.3: the essential oil of artimensia annua aerial parts

Table 2.4 Essential oils of artimensia annua are comprised of many constituents with the major compounding including (in relative % of the essential oil).

Table 3.1 Comparison of extraction efficiency

Table 9.1 Total purchased equipment cost

Table 9.2 Direct Cost

Table 9.3 Indirect cost

Table 9.4 Direct production cost

Table 9.5 Fixed charges and plant over head costs

Table 9.6 General expense cost

List of Figures

Figure 1.1 Chemical structure of artemisinin

Figure 2.1 Chemical structure of artemisinin and its derivatives

Figure 2.2 Artemisia annua plant leaves

Figure 3.1 Process flow sheet

Figure 4.1 Location of study area

Figure 4.2 Soxhelt extractor apparatus (Photo shoot: Author)

Figure 4.3 Rotary Evaporator (Photo shoot: Author)

List of Acronyms

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Acknowledgements

I would like to express my deep gratitude to my advisor Mr. Muhidin Seid (M.Sc) for his keen interest of valuable comments in the project work. I would like to thank the department head Chemical Engineering Mr. Solomon Workneh (M.Sc) for his assistance during the laboratory work in Addis Ababa

My thanks go to Dr. Mekuria Tadesse, Dr. Daniel Bisrat, Dr. Tesfaye and Ms. Foziya Ali from essential oil research center for their material support and laboratory work in the extraction and clean up of the crude extract.

Finally my thanks go to my families and colleagues for their motivation and encouragements during my stay in University. My father Mr. Yimer Abate without your unreserved assistance, encouragement, motive and kindness this work wouldn’t have reached its completion.

Abstract

Malaria disease is endemic in least developed countries like Ethiopia. The rapid development of drug-resistant malaria parasite strain leaves the need for new effective anti-malarial drugs. Artemisinin is a sesquiterpene lactone found in the leaves and flowers of plants Artemisia annua L and have different chemical structures and higher efficacy than others. The content of artemisinin is very small and from 0.5 to 1.2 % of dry weight of plants in Ethiopia. Extraction of artemisinin from Artemisia annua is mainly performed using hydrocarbon extraction processes. Extraction with Supercritical CO2, ethanol, ionic liquids, hydroflourocarbon HFC-134a and hexane extraction are extraction technologies and compared majorly in terms of their extraction efficiency, cost and drawback to environment. Since artemisinin is only present in the epidermis of the leaves, leaves were only washed on the outside by stirring appeared to be the most appropriate method and the first step in this research was solvent extraction using hexane. Step followed purify by repeated crystallization. Laboratory scale production data and procedures were listed for aremisinin extraction process. By using those data large scale production method was designed. And did material and energy balance calculations for large scale production process carried out by taking scientific approach followed by feasibility study. As a result my extractor was very large and it has 14.37m3 capacities it wasn’t found in market. So I designed the required larger extractor that should fulfill the target of the project i.e. with simple, rapid, cost effective, environmental friendly and practical method for the isolation of artemisinin from Artemisia annua. The design the extractor I used the optimum ratio between solvent and leaves, optimum extraction time, optimum extraction temperature and optimum design of the stirrer paddles. In these thesis work extractor design, skirt design, bolt design, reinforcement calculations, preliminary equipment design (sizing), economic analysis calculations were included. In the profitability analysis Return on investment, payback period and net present worth calculated and the project was checked its feasibility. The payback period is 1.22yrs and it is economically acceptable that means this market is promising in Ethiopia in addition to save millions of Ethiopian and African from killer malaria.

Key words: artemisinin, Artemisia annua L., sesquiterpene lactone, malaria, feasibility study, hexane extraction, material and energy balance calculations, extractor design

1. Introduction

1.1 Background

The history of herbal medicine is as old as human civilization. The widespread use of herbal remedies and healthcare preparations, as those described in ancient texts such as the Vedas and the Bible, and obtained from the commonly used traditional herbs and medicinal plants, has been traced to the occurrence of natural products with medicinal properties. World Health Organization (W.H.O.) currently encourages, recommends and promotes traditional/herbal remedies in National Health Care Programmers because such drugs are easily available at low cost, are comparatively safe and the people have faith in such remedies (Klayman, D.L., 1985).

Plants synthesize a tremendous number of secondary metabolites, in addition to compounds that are necessary for the growth and reproduction such as carbohydrates, proteins and lipids. As our understanding of chemistry and other natural sciences has increased, the active chemical compounds of the plants have been successfully isolated and identified. Extraction of crude drugs can be done by various processes depending on the physical nature of the drug and chemical properties of the constituents present in it. Various traditional methods used for the extraction of drugs include Infusion, Decoction, Digestion, Maceration and Percolation. Out of these Maceration and Percolation are of particular importance and most Pharmacopoeias refer to these processes for the extraction of crude drugs. The conventional extraction processes are time consuming, if use maceration, it will takes 2-7 days and involves bulk amount of solvents and ultimately there might be thermal decomposition of the target molecule. (Klayman D.L.,1985).

Artemisia annua is an aromatic annual herb traditionally grown in china as a medicinal plant for threatening different types of ailments including malaria, stomach complaints, and hemorrhoids. It has been successfully introduced in many African countries including Ethiopia; it is considered one of the most effective treatments against malaria in the world. It has become the source of income and a life-saver for the community. Actually this herb used for the crafting of aromatic wreaths, as a source of essential oils used in the beverages industry and also as a source of Artemisinin, a potent anti-malarial drug. Currently the plant grows in many regions as Australia, North- and South America, Southern Europe and Africa. Now farmers in Ethiopia are discovering its virtues (Ahmed Muzemil, 2008).

Frequently used extraction methods are liquid solvent extraction, Pressurized Solvent Extraction (PSE), Ultrasonic Assisted Extraction (UAE), Accelerated Solvent Extraction (ASE), Microwave-assisted Extraction (MAE), extractions with supercritical carbon dioxide (ScCO2), hydro fluorocarbon HFC-134a and ionic liquids. The empirical formula of Artemisinin is C15H22O5 (Dr. Al Fleming et al., 2007)

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Figure 1.1 Chemical structure of artemisinin (Malcolm Cutler et al., 2006)

The demand for new extraction techniques has encouraged the development of alternative extraction techniques and these techniques have enabled automation, shortened extraction time and reduced organic solvent consumption. In this study hexane extraction processes was applied.

1.2 Objective

1.2.1 General objective

The main objective of this project is extraction and isolation of artemisinin from the plant Artemisia annua, which have grown in Ethiopia with substantial quantity and purity, by hexane extraction process. And this study aims in evaluating the content of artemisinin and oil composition of Artemisia annua grown in Ethiopia.

1.2.2 Specific Objectives

- To extract Artemisia annua leaves using hexane
- To know oil yield and composition
- To do material and energy balance calculation
- To design large scale extractor
- To do economical feasibility calculations

1.3 Scope of Study

The plant Artemisia annua is the sole source of artemisinin in the industry. As a result it grows in most parts of Ethiopia; it becomes a source of income either by processed in Ethiopian industries or by export to abroad. In addition to its effectiveness on anti-malaria its effect will be assessed in anti-bacteria, anti-septic and anti-cancer properties and its constituent compounds will have analyzed by HPLC and GC/MS. Extract artemisinin from the different areas, latitudes, temperature and soil types of Ethiopia and choosing the optimum of these factors and grow the plant in large farms and further studying the properties of Artemisia annua plant to get a better amount of artemisinin content (Dr. Al Fleming et al., 2007).

The Chinese use it as food by make burger and salad (Malcolm Cutler et al., 2006). Develop this culture in Ethiopia consequently it plays significant role for the health and food security of the people.

2. Literature Review

2.1 Essential oil utilization and application

Essential oil is a concentrated, hydrophobic liquid containing volatile aroma compounds from plants. Essential oils are also known as volatile or ethereal oils or simply "oil off" the plant from which they were extracted. Oil is "essential" in the sense that it carries a distinctive scent, or essence, of the plant. Essential oils do not as a group needs to have any specific chemical properties in common, beyond conveying characteristic fragrances. An essential oil is a liquid that is generally distilled, most frequently by steam, from the leaves, stems, flowers, bark, roots, or other elements of a plant and they contain the true essence of the plant it was derived from (Malcolm Cutler et al., 2006).

Essential oils are generally extracted by distillation. Other processes include expression, or solvent extraction. They are used in perfumes, cosmetics, soap and other products, for flavoring food and drink, and for scenting incense and household cleaning products. Various essential oils have been used medicinally at different periods in history. Medical application proposed by those who sell medicinal oils range from skin treatments to remedies for cancer, and are often based on historical use of these oils for these purposes. Such claims are now subject to regulation in most countries, and have grown vaguer to stay within these regulations (Hsu, E., 2006).

Interest in essential oils has revived in recent decades with the popularity of aromatherapy, a branch of alternative medicine which claims that the specific aromas carried by essential oils have curative effects (Dr. Al Fleming et al., 2007).

2.2 History of Artemisia annua L.

Artemisia has been used by Chinese herbalists for more than a thousand years in the treatment of many illnesses, such as skin diseases and malaria. The earliest record dates back to 200 BC, in the "Fifty-two Prescriptions" unearthed from the Mawangdui Han Dynasty Tombs. Its anti-malarial application was first described in Zhouhou Beji Fang ("The Handbook of Prescriptions for Emergencies"), edited in the middle of the fourth century by Ge Hong. In the 1960s a research program was set up by the Chinese army to find an adequate treatment for malaria. In the early 1970s, Chinese scientists screening traditional Chinese medicinal herbs in the search for new anti-malarial isolated artemisinin from the herb known as Qing Hao (generally identified as Artemisia annua L. but may have originally referred to A. apiacea) (Hsu, E.,2006). In 1972, in the course of this research, Tu Youyou (Chinese) discovered artemisinin in the leaves of Artemisia annua. It was one of many candidates then remains tested by Chinese scientists from a list of nearly 200 traditional Chinese medicines for treating malaria. It was the only one that was effective, but it was found that it cleared malaria parasites from their bodies faster than any other drug in history. Artemisia annua is a common herb and has been found in many parts of the world. It remained largely unknown to the rest of the world for about ten years, until results were published in a Chinese medical journal. The report was met with skepticism at first, because the Chinese had made unsubstantiated claims about having found treatments for malaria before. (Hsu, E., 2006).

2.3 Artemisia Plant and Production

a. The Plant. Botanically, Artemisia annua is a vigorous weedy annual which is single-stemmed and ranges in height from one to two meters. It grows easily in temperate areas and tropical areas at higher altitudes and is raised in an increasing number of countries. It is well suited to both small-scale and plantation culture. The seed is extremely small and is usually grown to the seedling stage and transplanted. The best quality seed, in terms of production of leaves and yield of artemisinin, is provided by certain forms of purchased seed, which are generally limited in supply. Relatively few inputs are needed, aside from some fertilization, because the plants at present do not seem to have any particular insect or disease problems (this could change). Normally, some water is required to establish the crop and dry weather is needed at the harvest and for drying. Artemisinin levels of the plants tend to vary by variety, but the influences of area and growing conditions are not yet clear. It is principally planted early in the calendar year and needs five to six months to mature (Ferreira et al. 2005, WHO 2006).

b. Production in Africa. Production was initially largely limited to East Africa -Kenya, Tanzania, and Uganda - and was essentially tied to the activities of one holding company, Advanced Bio-Extracts Ltd (ABE) (www.abextracts.com), and two subsidiaries: East African Botanicals (EAB), Ltd. in Kenya and African Artemisia Ltd. (AA) in Tanzania. In 2007, the name of the firm, reflecting a new investor, was changed to Botanical Extracts Ltd. (BEEPZ). Contract production was utilized and the firm supplied seed that has proven well adapted to the region; this process also provided a relatively uniform level of artemisinin. The increased demand for artemisinin, starting in 2004, stimulated the efforts to increase the production of Artemisia in East Africa. ABE clearly was in a position to do so. The area subsequently placed under various production arrangements (leased or joint venture efforts) in Kenya, Uganda, and Tanzania (north) expanded to approximately 1,650 ha. (4,100 acres) in 2005. Most of the planting for the calendar year (58.4%) was carried out in the second quarter, followed by lesser amounts in the third (23.8%) and fourth quarters (17.7%). The planted area was principally in Kenya (nearly 65%) followed by Uganda (19%) and Tanzania (north, over 16%) (TechnoServe 2005). Both small and large farms were involved. In 2006, 7,500 farmers were reportedly involved, but area was not revealed. Area estimates for 2007 ranged from 3,500 to 4,000 ha. (8,650 to 9,900 acres). (Pilloy 2008). Currently the plant widely cultivating in many places of Ethiopia.

2.4 Uses of Artemisia annua L.

2.4.1 Medicinal Uses

Artemisia annua essential oil is beneficial for anti-bacteria, anti-periodic, anti-septic and anti-cancer properties. It has the following medicinal uses

- Parasite treatment: It is commonly used in tropical nations which can afford it, preferentially as part of a combination-cocktail with other anti malarias in order to prevent the development of parasite resistance (Keasling JD, et al ., 2006).

- Malaria treatment: Artemisinin is an unusual sesquiterpene lactone incorporating an endoperoxide group. It has potent anti-plasmodial activity and was shown in clinical trials to be highly effective against malaria, including patients with cerebral malaria and patients with malaria parasites resistant to chloroquine (Klayman, D.L., 1985). In contrast to other antimalarials such as quinine, (used for treating chloroquine-resistant malaria), artemisinin was found to be remarkably non-toxic and the importance of its discovery against the background of a rising incidence of multidrug-resistant malaria parasites cannot be underestimated. Several semi-synthetic artemisinin derivatives including artemether, arteether and sodium artesunate are now in clinical use worldwide for the treatment of malaria (Olumnese P., 2006). However, antimalarial drugs such as the artemisinin derivatives are expensive and are not accessible to the majority of people who are at risk of malaria, especially in Africa with the result that approximately 1 million people, mostly children, die from malaria each year (Kokwaro, 2007).

- Cancer treatment: The plant has also been shown to have anti-cancer properties. It is said to have the ability to be selectively toxic to some breast cancer cells and some form of prostate cancer, there have been exciting preclinical results against leukemia, and other cancer cells. Artemisinin is the active component of the herb Artemisia annua. (www.allergyresearchgroup.com) The Artemisinin extract provides approximately 300 times more of the active ingredient than the whole herb itself. Artemisinin is non-toxic, so treatment can continue indefinitely with no expected side effects. The World Health Organization recommends Artemisinin in the treatment of Malaria in areas where resistance to Malaria drugs has developed. Artemisinin has a unique chemical structure that prevents cancer resistance. The active molecule in artemisinin reacts with free iron, releasing highly-reactive free radicals that destroy cells harboring free iron. (The iron in our red blood cells is not free iron. Rather, it is strongly bonded to resist the effects of Artemisinin.) Most cancer cells have high rates of iron intake, due to their greedy appetite for blood to support their rapid growth. Thus, if cancer developed resistance to Artemisinin, its blood supply would diminish resulting in tumor death (www.allergyresearchgroup.com).

2.4.2 Edible Uses

In modern-day central China, specifically Hubei Province, the stems of this plant are used as food in a salad-like form. The food product "cold-mixed wormwood" is a slightly bitter salad with strong acid overtones from the spiced rice vinegar used as a marinade. It is considered a delicacy and is typically more expensive to buy than meat. It is also used as flavorings. (Hsu, E., 2006).

2.5 Chemical Constituents of Artemisia Annua

Several secondary metabolites characterize the chemical composition of the genus Artemisia. According to surveyed literature, almost all classes of compounds are observed to be present in the genus with particular reference to terpenoids and flavonoids. A large number of monoterpenoid compounds have been characterized from the essential oil of Artemisia annua .The yield of the oil generally varies between 0.3% and 0.4% (v/w). The chemical constituents of Artemisia annua oil varied from country to country for example principal of Chinese oil were 63.9% Artemisia Ketone, 7.5% Artemisia alcohol 5.1%, 4.7% guaine and 3.3% comphor. The Vietnamese oil contained camphor (21.8%), germacerene D (18.3%) and 1.8-cineole (3.1%) etc, in this plant were given much attention is artemisinin, which is responsible for anti-malarial activity. Artemisinin is by lack of nitrogen containing heterocyclic ring system which is found in most anti-malarial drugs. A large number of flavonoids have been reported from the plant and also few compounds like triterpenoids and steroids have been reported (Ahmed Muzemil, 2008).

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Figure 2.1 Chemical structures of artemisinin and its derivatives (Ahmed Muzemil, 2008).

A large variation in artemisinin content has been observed in the leaves of different samples of Artemisia annua. Contents varying from 0.01 to 1.38% in the dry leaves have been reported (table1). The variation in the content of artemisinin might be because of several reasons. In addition to the use of different methods for extraction and analysis, the time of collection and preparation of the samples, contributes a lot to the variation among different samples. Moreover, an environmental factor such as temperature and availability of nutrient has also much to do with the variations (Dr. Al Fleming et al., 2007).

Table 2.1 Artemisinin content in different samples of A. annua % dry weight (Malcolm Cutler et al., 2006).

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2.6 Physical and Chemical properties of Artemisia Annua

2.6.1 Physical Properties of Artemisia annua

It has fern-like leaves, bright yellow flowers, and a camphor-like scent. Its height averages about 2 m tall, and the plant has a single stem, alternating branches, and alternating leaves which range 2.5 cm in length. It is in flower from august to September, and the seeds ripen from September to October (Abebe D. et al, 1993).

Table 2.2 scientific classifications (www.wikipedia.com).

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Figure 2.2 Artemisia annua plant leaves (Dr. Al Fleming, 2007).

Physical data

Density --- 1.24 ± 0.1 g/cm³

Melt. Point --- 152–157 °C (306–315 °F)

Chemical data

Mol. Mass --- 282.332 g/mol

Formula --- C15H22O5 (www.wikipedia.com).

2.6.3 Physico-Chemical Properties

Artemisinin is low thermal and chemical stability of the endoperroxide function, low polarity and hence poor solubility in water and good solubility in organic solvents, its extraction with non-polar solvents (Malcolm Cutler et al, 2006).

2.6.4 Over View of Artemisinin Oil

Artemisinin is a drug used to treat multi-drug resistant strains of falciparum malaria. The compound (a sesquiterpene lactone) is isolated from the plant Artemisia annua. Not all plants of this species contain artemisinin. It can be synthesized from artemisinic acid. Use of the drug by itself as a monotherapy is explicitly discouraged by the World Health Organization as there have been signs that malarial parasites are developing resistance to the drug. The drug is also being studied as a treatment for cancer (Wright, 2002).

2.7 Composition of Artemisinin

Table 2.3: the essential oil of artimensia annua aerial parts (Ahmed Muzemil, 2008)

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Table 2.4 Essential oils of artimensia annua are comprised of many constituents with the major compounding including (in relative % of the essential oil) (Ahmed Muzemil, 2008)

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Benefits of Artemisinin

- Health, societal and economic Benefit
- Environmental Benefit
- Employment Benefit: local economies benefit from job creation in agricultural and manufacturing sectors (Malcolm Cutler et al., 2006)

2.8 Market Assessment

Ethiopia have imported medicines from abroad that are made from Artemisia annua with expensive prices, but Artemisia annua and other natural medicines plants have been growing in the country. The reason for entering of extraction of this plant at this time is to produce artemisinin in large, for the country due to this significant amount of money reduces and decrease the death rate of people by malaria.

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